U.S. patent application number 12/856765 was filed with the patent office on 2010-12-09 for method for producing hydrogen sulphide and the use thereof, in particular, for depolluting heavy metal-containing flows.
This patent application is currently assigned to INSTITUT DE RECHERCHE POUR LE DEVELOPPEMENT (IRD). Invention is credited to Bruno Chardin, Yannick Aman Baptiste Combet-Blanc, Marie-Laure Fardeau, Bernard Marcel Noel Ollivier.
Application Number | 20100307985 12/856765 |
Document ID | / |
Family ID | 35448071 |
Filed Date | 2010-12-09 |
United States Patent
Application |
20100307985 |
Kind Code |
A1 |
Ollivier; Bernard Marcel Noel ;
et al. |
December 9, 2010 |
METHOD FOR PRODUCING HYDROGEN SULPHIDE AND THE USE THEREOF, IN
PARTICULAR, FOR DEPOLLUTING HEAVY METAL-CONTAINING FLOWS
Abstract
A process for the decontamination of an effluent containing one
or more dissolved metals is provided. The process includes
producing hydrogen sulphide in an aqueous medium by culturing
alkaliphilic sulphate-reducing or thio-sulphate reducing bacteria
in the presence of an organic compound serving as an electron donor
and in the presence of a sulphurous compound serving as an electron
acceptor. The effluent is contacted with the hydrogen sulphide and
the dissolved metals are reduced and/or precipitated in the form of
metal sulphides.
Inventors: |
Ollivier; Bernard Marcel Noel;
(Roquevaire, FR) ; Combet-Blanc; Yannick Aman
Baptiste; (Marseille, FR) ; Fardeau; Marie-Laure;
(Les Pennes-Mirabeau, FR) ; Chardin; Bruno;
(Aubagne, FR) |
Correspondence
Address: |
YOUNG & THOMPSON
209 Madison Street, Suite 500
Alexandria
VA
22314
US
|
Assignee: |
INSTITUT DE RECHERCHE POUR LE
DEVELOPPEMENT (IRD)
Paris Cedex
FR
|
Family ID: |
35448071 |
Appl. No.: |
12/856765 |
Filed: |
August 16, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11919517 |
Dec 13, 2007 |
7799222 |
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PCT/FR2006/000954 |
Apr 27, 2006 |
|
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12856765 |
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Current U.S.
Class: |
210/716 |
Current CPC
Class: |
C02F 2101/101 20130101;
C02F 9/00 20130101; C02F 2101/20 20130101; C02F 3/345 20130101;
Y10S 210/912 20130101; C02F 3/34 20130101; C02F 1/5236 20130101;
C12P 3/00 20130101; C02F 2101/22 20130101; C02F 9/00 20130101; C02F
2101/101 20130101; C02F 3/34 20130101; C02F 1/5236 20130101; C02F
2101/20 20130101; C02F 2101/22 20130101 |
Class at
Publication: |
210/716 |
International
Class: |
C02F 1/52 20060101
C02F001/52 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 29, 2005 |
FR |
0504386 |
Claims
1. A process for the decontamination of an effluent containing one
or more dissolved metals, the process comprising: (i) producing
hydrogen sulphide in a form largely soluble in an aqueous medium
comprising the following stages: a stage of culturing alkaliphilic
sulphate-reducing or thio-sulphate reducing bacteria selected from
at least one species of the Desulfohalobiaceae family or of the
Desulfonatronum genus, or of which the gene coding for the
ribosomal RNA 16 S exhibits a homology of at least 97% with the
corresponding gene of any one of the species of the
Desulfohalobiaceae family or of the Desulfonatronum genus, in the
presence of an organic compound serving as an electron donor, and
in the presence of a sulphurous compound serving as an electron
acceptor, which leads to the formation of hydrogen sulphide, and a
sequence of the following two stages: a first stage in which the
culturing of the bacteria is carried out under conditions
appropriate for the growth of the bacteria and for the concomitant
production of hydrogen sulphide, and a second stage in which the
culturing of the bacteria is carried out under conditions
appropriate for the production of hydrogen sulphide in the absence
of growth of the said bacteria, and optionally repeating the
sequence of the stages constituting one cycle several times if
necessary; (ii) contacting the effluent with the hydrogen sulphide;
and (iii) reducing the one or more dissolved metals and/or
precipitating the one or more dissolved metals in the form of metal
sulphides.
2. The process according to claim 1, wherein said contacting the
effluent with the hydrogen sulphide is carried out at a pH in a
range of from 2 to 12.
3. The process according to claim 1, wherein said contacting the
effluent with the hydrogen sulphide is carried out at a pH in a
range of from about 7 to 12.
4. The process according to claim 1, wherein producing the hydrogen
sulphide is carried out in a culturing tank, and contacting the
effluent with the hydrogen sulphide is carried out in a reaction
tank, the culturing tank and the reaction tank being separate
tanks, and between the producing hydrogen sulphide step and the
contacting the effluent with the hydrogen sulphide step, the
process further comprises injecting all or part of the hydrogen
sulphide produced in the culturing tank into the reaction tank.
5. The process according to claim 4, wherein no fraction of the
effluent is injected into the culturing tank.
6. The process according to claim 4, wherein a fraction of the
effluent is injected into the culturing tank.
7. The process according to claim 4, wherein contacting the
effluent with the hydrogen sulphide in the reaction tank is carried
out in the absence of a gaseous phase and in such a manner that
there is no release of gaseous hydrogen sulphide.
8. The process according to claim 4, further comprising: measuring
the concentration of hydrogen sulphide produced in the culturing
tank; estimating the concentration of the one or more dissolved
metals in the effluent to be decontaminated; and adjusting the
quantity of hydrogen sulphide to be injected into the reaction tank
based on the result of said measuring and of said estimating.
9. The process according to claim 1, wherein the one or more
dissolved metals is selected from the group consisting of copper,
zinc, arsenic, cadmium, chromium, tin, manganese, mercury, nickel,
and lead.
10. The process according to claim 1, wherein the organic compound
serving as an electron donor is formate and the sulphurous compound
serving as an electron acceptor is thiosulphate.
11. The process according to claim 1, wherein the culturing of the
alkaliphilic sulphate-reducing or thiosulphate-reducing bacteria is
carried out at a pH greater than or equal to 9.
12. The process according to claim 1, wherein the culturing of the
alkaliphilic sulphate-reducing or thiosulphate-reducing bacteria is
carried out at a pH greater than or equal to 9.5.
13. The process according to claim 1, wherein the culturing of the
alkaliphilic sulphate-reducing or thiosulphate-reducing bacteria is
carried out at a pH greater than or equal to 10.
14. The process according to claim 1, wherein the bacteria have a
tolerance to hydrogen sulphide, and the bacteria tolerate
concentrations of hydrogen sulphide at least greater than 20
mM.
15. The process according to claim 1, wherein the bacteria belong
to the species Desulfonatronum lacustre.
16. The process according to claim 1, wherein the bacteria belong
to the species Desulfonatronovibrio hydrogenevorans.
17. The process according to claim 1, wherein the culturing is
carried out on a support suitable for the growth of the bacteria,
leading to the formation of a biofilm.
18. The process according to claim 1, wherein the passage from one
stage to the other for producing hydrogen sulphide is carried out
by addition of one or several acidic or basic chemical substances
resulting in a change in the pH of the culture medium of the
bacteria.
19. The process according to claim 1, wherein the first stage is
carried out at a pH ranging from 9 to 10 and the second stage is
carried out at a pH greater than 10.
20. The process according to claim 1, wherein the hydrogen sulphide
is produced at a concentration greater than or equal to 10 mM.
21. The process according to claim 1, wherein the hydrogen sulphide
is produced at a concentration greater than or equal to 20 mM.
22. The process according to claim 1, wherein the hydrogen sulphide
is produced at a concentration greater than or equal to 30 mM.
23. The process according to claim 1, wherein the bacteria belong
to the species Desulfonatronum lacustre, the organic compound
serving as an electron donor is formate, the sulphurous compound
serving as an electron acceptor is thiosulphate and the bacteria
are continuously cultured on a biofilm at a pH greater than 10.
Description
[0001] This application is a divisional of co-pending application
Ser. No. 11/919,517 filed on Dec. 13, 2007, which is the 35 U.S.C.
.sctn.371 national stage of International PCT/FR2006/000954 filed
on Apr. 27, 2006, which claims priority to French Application No.
0504386 filed on Apr. 29, 2005. The entire contents of each of the
above-identified applications are hereby incorporated by
reference.
[0002] The invention relates to a process for the production of
hydrogen sulphide and the use thereof, in particular for the
decontamination of effluents containing heavy metals.
[0003] Heavy metals can be highly toxic to man and his environment.
These heavy metals are not biodegradable, and hence they are of a
cumulative nature. Thus more and more restrictive discharge
standards have been imposed on industrial activities discharging
metals.
[0004] The processes most commonly used to separate the heavy
metals contained in industrial effluents utilise the formation of
metal hydroxides. However, these processes do not always satisfy
the current environmental standards relating to the acceptable
levels of dissolved metals in effluents.
[0005] Another technology already in use involves the formation of
metal sulphides. This requires the use of expensive synthetic
polysulphides or the formation of gaseous hydrogen sulphide by the
action of hydrochloric acid on sodium sulphide.
[0006] Biological alternatives to this mode of treatment, again
involving the production of gaseous hydrogen sulphide, have been
proposed in the past. These biological alternatives are based on
two main types of biological mechanisms capable of leading to the
formation of hydrogen sulphide: [0007] 1) The assimilative
reduction of sulphate is an anabolic function which allows the
majority of bacteria, fungi and plants to incorporate sulphur into
amino acids (cysteine, methionine and cystine), vitamins (thiamine
and biotin) and other sulphurous molecules (ferredoxin for example
. . . ) present in the cells of these organisms. This reduction
never leads directly to the production of hydrogen sulphide.
Nonetheless, the latter is released indirectly during the
fermentation of the proteinaceous organic matter. [0008] 2) The
dissimilative reduction of sulphates is carried out by
sulphate-reducing bacteria. In this anaerobic respiratory process,
the sulphate is used as a terminal electron acceptor during the
oxidation of hydrogen or of reduced organic compounds such as
acetate and propionate. During this metabolism, the substrates are
most often partially oxidised to acetate or, in some cases, totally
oxidised resulting in the formation of CO.sub.2.
[0009] Many patents or patent applications relate to the use of
sulphate-reducing bacteria for the precipitation of metal ions as
metal sulphides, in order to decontaminate effluents such as mine
effluents or waste waters.
[0010] Thus, the documents WO 80/02281 and U.S. Pat. No. 4,522,723,
relate to the use of bacteria of the Desulfovibrio or
Desulfotomaculum type to reduce the levels of heavy metals in
effluents. The document U.S. Pat. No. 4,108,722 envisages the
injection of Vibrio and Desulfovibrio bacteria into contaminated
subterranean aquifer reservoirs. The document U.S. Pat. No.
5,062,956 relates more specifically to hexavalent chromium
treatment. Three other documents, U.S. Pat. No. 5,587,079, WO
97/29055 and WO 02/06540 propose other processes for biological
precipitation of metals, with the distinctive feature that the
precipitation is carried out sequentially, by varying the pH
(mainly between 2.5 and 6.5) in order for example first to
precipitate copper, then zinc etc. . . . The document WO 97/05237,
which discloses the use of methylotrophic bacteria, i.e. which are
capable of using methanol as the sole source of carbon, may also be
cited. It must also be noted that systems of co-culture of bacteria
of different strains have also been envisaged, for example in the
document U.S. Pat. No. 4,789,478 or the document EP 0 692 458.
[0011] A disadvantage of the processes cited above resides in the
fact that sometimes considerable quantities of hydrogen sulphide in
gaseous form (H.sub.2S) are produced during these processes. This
is particularly the case when it is desired to obtain maximal
precipitation of the metals initially dissolved in the effluent to
be decontaminated, which requires the use of the hydrogen sulphide
in excess. Now hydrogen sulphide in gaseous form is toxic,
corrosive, harmful to the environment and requires an appropriate
supplementary restrictive treatment.
[0012] One way of avoiding this disadvantage consists in producing
dissolved hydrogen sulphide (in the form of HS.sup.-) instead of
gaseous hydrogen sulphide. In order to do this, it is desirable
that the pH of the culture solution in which the sulphide is
produced by the sulphate-reducing bacteria be as high (basic) as
possible. Now, in the great majority of the sulphate-reducing
bacteria the use of a high pH adversely affects the sulphide
production yield and can even be lethal to the culture.
[0013] One of the aspects of the invention is to produce
essentially soluble hydrogen sulphide, in good yield, by means of
sulphate- or thiosulphate-reducing bacteria.
[0014] One of the other aspects of the invention is to propose new
culture conditions for certain sulphate-reducing bacteria, making
it possible in particular to make use of their
thiosulphate-reducing properties.
[0015] One of the other aspects of the invention is to propose a
process for the decontamination of effluents containing heavy
metals by means of hydrogen sulphide produced by the culturing of
sulphate- or thiosulphate-reducing bacteria.
[0016] Yet another aspect of the invention is to produce hydrogen
sulphide for the decontamination of effluents containing heavy
metals, while avoiding the undesirable presence of gaseous hydrogen
sulphide.
[0017] These different aspects are obtained by using certain
recently discovered sulphate-reducing alkaliphilic bacteria, not
until now used to produce hydrogen sulphide or a fortiori for the
decontamination of effluents laden with metals.
[0018] The invention thus relates to the use of alkaliphilic
sulphate-reducing or thiosulphate-reducing bacteria selected from
at least one species of the Desulfohalobiaceae family or of the
Desulfonatronum genus or of which the gene coding for the ribosomal
RNA 16 S exhibits a homology of at least 97% with the corresponding
gene of any one of the species of the Desulfohalobiaceae family or
of the Desulfonatronum genus, to produce hydrogen sulphide in a
form largely soluble in an aqueous medium.
[0019] By "alkaliphilic bacteria" is meant bacteria whose life,
growth, and various metabolic and enzymatic activities preferably
take place at a basic pH.
[0020] A taxonomic, morphological and physiological description of
the bacteria used in the invention has been given in the following
articles: [0021] Pikuta et al., Desulfonatronum lacustre gen. nov.
sp. nov.: a new alkaliphilic sulphate-reducing bacterium utilizing
ethanol; Microbiology 67, 105; [0022] Zhilina et al.,
Desulfonatronovibrio hydrogenovorans gen. nov. sp. nov., an
alkaliphilic, sulphate-reducing bacterium; Int. J. Syst. Bacteriol.
January 1997, p. 144; [0023] Pikuta et al., Desulfonatronum
thiodismutans sp. nov., a novel alkaliphilic, sulphate-reducing
bacterium capable of lithoautotrophic growth; Int. J. Syst. Evol.
Microbiol. 53, 1327.
[0024] "Homology" is used to designate the proportion of identity
between two nucleic acid sequences. This homology can be measured
by seeking to align the said sequences using an algorithm such as
that defined in Altschul et al. (Nucl. Acid Res. 25:3389, 1997) or
by using for example the software Clustal W, well known to the
person skilled in the art and described in Thompson et al. (Nucl.
Acid Res. 22:4673, 1994).
[0025] By "form largely soluble" is meant a ratio of soluble
hydrogen sulphide produced to gaseous hydrogen sulphide produced
greater than 1, and in particular greater than 100.
[0026] As will be described in detail below, for the conditions for
culturing the sulphate-reducing bacteria according to the
invention, novel substrates, namely formate and thiosulphate, are
preferably used. The formate serves as an energy source, while the
thiosulphate serves as an electron acceptor and source of sulphur:
the strains of bacteria used in the invention are thus
thiosulphate-reducing as well as being sulphate-reducing. This
property of certain sulphate-reducing bacteria of utilising
thiosulphate as a substrate has not been used in the previously
cited processes for metal decontamination.
[0027] The use of alkaliphilic sulphate- or thiosulphate-reducing
bacteria under the conditions of the invention makes it possible to
produce hydrogen sulphide in a very largely soluble form, and in an
exceptionally high yield.
[0028] Another decisive advantage of the invention is the
possibility that it affords of working with a pure culture pure
without having to perform any sterilisation, which makes
considerable savings possible. In fact, the particular culture
conditions which are used (high pH, mineral content, absence of
O.sub.2, high concentration of hydrogen sulphide . . . ) ensure a
strong selection pressure.
[0029] Advantageously, the use of alkaliphilic sulphate-reducing or
thiosulphate-reducing bacteria according to the invention is
carried out at a pH greater than or equal to about 9, in particular
at a pH greater than or equal to about 9.5, in particular at a pH
greater than or equal to about 10.
[0030] The proportion of the hydrogen sulphide produced according
to the invention which is in gaseous form (in other words the ratio
of gaseous hydrogen sulphide produced to the dissolved hydrogen
sulphide produced) in fact depends on the pH at which the hydrogen
sulphide is produced. At an acidic pH, the predominant form of the
hydrogen sulphide is the gaseous form. At a pH of 7 there are about
as many molecules of H.sub.2S as HS.sup.- ions. At a pH of 9, the
proportion of gaseous hydrogen sulphide is only about 1/100. At a
pH of 9.5, the proportion of gaseous hydrogen sulphide is only
about 1/500. At a pH of 10, the proportion of gaseous hydrogen
sulphide is only about 1/1000.
[0031] According to another advantageous implementation of the
invention, the use of alkaliphilic sulphate-reducing or
thiosulphate-reducing bacteria takes place in the form of culturing
in the presence of an organic compound serving as an electron
donor, in particular formate, and in the presence of a sulphurous
compound serving as an electron acceptor, in particular
thiosulphate.
[0032] However, it is important to note that, depending on the
bacterial strains utilised, other substrates can serve for the
production of sulphide: for example sulphate, sulphite or sulphur
as electron acceptor, and ethanol or dihydrogen as electron
donor.
[0033] However that may be, the production of hydrogen sulphide is
carried out in the invention via the reduction of a sulphurous
compound. In the case where this sulphurous compound is
thiosulphate (S.sub.2O.sub.3.sup.2-), the enzymatic reduction
mechanisms involved are in particular based on thiosulphate sulphur
transferase (or rhodanese) and on thiosulphate reductase.
[0034] In the first case, the outcome of the thiosulphate reduction
reaction is equivalent to an oxidation of a thiol group by the
thiosulphate, the sulphonyl part of which is reduced to
sulphite:
S.sub.2O.sub.3.sup.2-+2RS.sup.-+H.sup.+.fwdarw.SO.sub.3.sup.2-+R--SS--R+-
HS.sup.-
[0035] In the second case, the thiosulphate is cleaved to sulphite
and sulphide, and the sulphite is then reduced to sulphide, with
the final outcome:
S.sub.2O.sub.3.sup.2-+4H.sub.2.fwdarw.2 HS.sup.-+3H.sub.2O
[0036] Also a subject of the invention is a process for the
production of hydrogen sulphide in a form largely soluble in an
aqueous medium comprising: [0037] a stage of culturing alkaliphilic
sulphate-reducing or thiosulphate-reducing bacteria selected from
at least one species of the Desulfohalobiaceae family or of the
Desulfonatronum genus or of which the gene coding for the ribosomal
RNA 16 S exhibits a homology of at least 97% with the corresponding
gene of any one of the species of the Desulfohalobiaceae family or
of the Desulfonatronum genus, in the presence of an organic
compound serving as an electron donor, in particular formate, and
in the presence of a sulphurous compound serving as an electron
acceptor, in particular thiosulphate, which leads to the formation
of hydrogen sulphide.
[0038] This bacterial culturing stage can for example be carried
out in a standard reactor such as those available on the market for
fermentation, on the pilot scale and on the industrial scale
alike.
[0039] Advantageously, in the process for the production of
hydrogen sulphide according to the invention, the culturing of the
alkaliphilic sulphate-reducing or thiosulphate-reducing bacteria is
carried out at a pH greater than or equal to about 9, in particular
at a pH greater than or equal to about 9.5, in particular at a pH
greater than or equal to about 10.
[0040] If necessary, the pH can be measured and regulated by the
addition of acidic and/or basic substances.
[0041] According to a preferred implementation of the process for
the production of hydrogen sulphide according to the invention, the
bacteria are selected such that they display a tolerance to
hydrogen sulphide, the said tolerance being characterised in that
the bacteria tolerate concentrations of hydrogen sulphide at least
greater than 20 mM.
[0042] By "tolerance" is meant the survival of the bacteria and the
maintenance of normal metabolic activity, which can be observed by
the consumption of the energy source.
[0043] This good tolerance of the bacteria in culture makes it
possible to obtain solutions with a high concentration of hydrogen
sulphide.
[0044] Advantageously, the bacteria used in the process for the
production of hydrogen sulphide according to the invention belong
to the species Desulfonatronum lacustre.
[0045] Advantageously, the bacteria used in the process for the
production of hydrogen sulphide according to the invention belong
to the species Desulfonatronovibrio hydrogenevorans.
[0046] According to an advantageous implementation of the
invention, the culturing involved in the process for the production
of hydrogen sulphide is carried out on a support suitable for the
growth of the bacteria, leading to the formation of a biofilm.
[0047] By "biofilm" is meant bacteria preferentially immobilised on
a suitable support, of the pozzolane, Cloisonyl.RTM., Bio-Net.RTM.
or Sessil.RTM. type for example, instead of a simple suspension of
bacteria in solution. Culturing in biofilms makes it possible to
obtain high concentrations of bacterial cells locally and to
produce hydrogen sulphide more rapidly. Moreover, few bacterial
cells are removed from the reactor during the withdrawal of the
culture solution containing the hydrogen sulphide, since the
bacteria are largely not in suspension.
[0048] According to a preferred implementation of the process for
the production of hydrogen sulphide according to the invention,
this comprises a sequence of the following two stages: [0049] a
first stage in which the culturing of the bacteria is carried out
under conditions appropriate for the growth of the said bacteria
and for the concomitant production of hydrogen sulphide, and [0050]
a second stage in which the culturing of the bacteria is carried
out under conditions appropriate for the production of hydrogen
sulphide in the absence of growth of the said bacteria,
[0051] it being possible to repeat several times the sequence of
the said stages constituting one cycle if necessary.
[0052] By "growth of the said bacteria" is meant in particular
their multiplication by cell division.
[0053] By "production of hydrogen sulphide in the absence of growth
of the said bacteria" is meant on the one hand the substantial
arrest or marked slowing of growth, in other words of the
multiplication of the bacteria, but also on the other hand the
survival of these bacteria or at least the maintenance of enzymatic
activity of bacterial origin causing the formation of hydrogen
sulphide in the medium.
[0054] Preferably, the passage from one of the said stages to the
other is in particular carried out by means of addition of one or
several acidic or basic chemical substances, causing a change in
the pH of the bacterial culture medium.
[0055] Particularly preferably, the said first stage is carried out
at a pH ranging from about 9 to about 10 and the said second stage
is carried out at a pH greater than about 10.
[0056] In fact, the bacteria used in the invention are capable of
maintaining their hydrogen sulphide production activity at
particularly extreme alkaline pH, that is to say in particular at a
pH greater than about 10. It is thus advantageous to culture the
bacteria of the invention in an initial period at their optimal
culture pH, which in general lies between about 9 and about 10, so
as to obtain the greatest possible number of bacteria, then in a
second period to pass to a pH greater than about 10 so as to
continue to produce hydrogen sulphide, but under chemical
conditions of the medium such that the ratio of the gaseous
hydrogen sulphide produced to hydrogen sulphide produced is as low
as possible, and in particular less than 1/100.
[0057] According to a preferred implementation of the invention,
the hydrogen sulphide is produced at a concentration greater than
or equal to about 10 mM, in particular at a concentration greater
than or equal to about 20 mM, in particular at a concentration
greater than or equal to about 30 mM, in particular at a
concentration greater than or equal to about 40 mM.
[0058] The typical specific rate of production of hydrogen sulphide
that can be attained according to the invention is at least 2.9
mmol HS.sup.- g.sup.-1 hr.sup.-1 and can range up to 5 mmol
HS.sup.- g.sup.-1 hr.sup.-1.
[0059] According to another preferred implementation of the
invention, the hydrogen sulphide is produced by a culture of
bacteria belonging to the species Desulfonatronum lacustre, the
organic compound serving as an electron donor being formate, and
the sulphurous compound serving as an electron acceptor being
thiosulphate, and the bacteria are continuously cultured on a
biofilm at a pH greater than about 10.
[0060] Also a subject of the invention is a process for the
decontamination of an effluent containing one or more dissolved
metals comprising: [0061] a stage of production of hydrogen
sulphide in a form largely soluble in an aqueous medium by means of
a culture of alkaliphilic sulphate-reducing or
thiosulphate-reducing bacteria selected from at least one species
of the Desulfohalobiaceae family or of the Desulfonatronum genus or
of which the gene coding for the ribosomal RNA 16 S exhibits a
homology of at least 97% with the corresponding gene of any one of
the species of the Desulfohalobiaceae family or of the
Desulfonatronum genus in the presence of an organic compound
serving as an electron donor, in particular formate, and in the
presence of a sulphurous compound serving as an electron acceptor,
in particular thiosulphate, and [0062] a stage of contacting the
said effluent with the hydrogen sulphide obtained in the preceding
stage, resulting in the reduction of the said dissolved metals
and/or the precipitation of the said dissolved metals in the form
of metal sulphides, the said contacting being carried out at a pH
ranging from about 2 to about 12.
[0063] By "decontamination of an effluent containing one or more
dissolved metals" is meant the significant reduction of the
concentration of one or more metals dissolved in the effluent, and
in particular a reduction below the thresholds imposed by the
various environmental standards for discharges.
[0064] Examples of thresholds for metal concentrations in liquid
discharges currently imposed by French legislation are as follows:
0.5 mg/L for copper; 0.5 to 2 mg/L for zinc; 0.05 to 0.5 mg/L for
arsenic; 0.05 to 0.2 mg/L for cadmium; 0.1 mg/L for hexavalent
chromium; 0.5 to 2 mg/L for tin; 0.03 to 0.05 mg/L for mercury; 0.5
to 2 mg/L for nickel and 0.1 to 0.5 mg/L for lead. It must also be
noted that the thresholds can vary depending on the industries
involved, and that bylaws sometimes locally set thresholds up to
100 times lower than the values cited above.
[0065] Now, with the exception of the special case of chromium,
almost all the metals can be precipitated as metal sulphides by the
action of hydrogen sulphide and display lower solubility in the
form of metal sulphides than in the form of metal hydroxides.
[0066] In fact the minimal solubilities observed, at various pH,
for the metal hydroxides and sulphides are as follows: [0067]
5.2.times.10.sup.-2 mg/L for arsenic sulphide (and no formation of
arsenic hydroxide); [0068] 6.7.times.10.sup.-10 mg/L for cadmium
sulphide versus 2.3.times.10.sup.-5 mg/L for cadmium hydroxide;
[0069] 1.0.times.10.sup.-8 mg/L for copper sulphide versus
2.2.times.10.sup.-2 mg/L for copper hydroxide; [0070]
3.8.times.10.sup.-8 mg/L for tin sulphide versus
1.1.times.10.sup.-4 mg/L for tin hydroxide; [0071]
2.1.times.10.sup.-3 mg/L for manganese sulphide versus 1.2 mg/L for
manganese hydroxide; [0072] 9.0.times.10.sup.-20 mg/L for mercury
sulphide versus 3.9.times.10.sup.-4 mg/L for mercury hydroxide;
[0073] 6.9.times.10.sup.-8 mg/L for nickel sulphide versus
6.9.times.10.sup.-3 mg/L for nickel hydroxide; [0074]
3.8.times.10.sup.-9 mg/L for lead sulphide versus 2.1 mg/L for lead
hydroxide; and [0075] 2.3.times.10.sup.-7 mg/L for zinc sulphide
versus 1.1 mg/L for zinc hydroxide;
[0076] In the particular case of chromium in its hexavalent form,
precipitation in the form of sulphide is not possible, but on the
other hand the hydrogen sulphide makes it possible to reduce the
Cr.sup.6+ ion to the Cr.sup.3+ ion (Kim et al. 2001, Chromium VI
reduction by hydrogen sulfide in aqueous media: stoichiometry and
kinetics. Environ. Sci. Technol. 35(11): 2219-2225), which is much
less toxic and less soluble, particularly in its hydroxide
form.
[0077] Further, the solubility of the metal hydroxides is generally
minimal for a certain optimal pH value, whereas the solubility of
the metal sulphides is typically a purely decreasing function of
the pH, so that it is always advantageous, solely from the point of
view of the solubility of the sulphides, to work at a pH as basic
as possible.
[0078] In summary, the decontamination of effluents according to
the invention is thus particularly advantageous compared to
processes making use of a simple precipitation in the form of
hydroxides for: [0079] arsenic, which cannot be insolubilised as
hydroxide, and whose insolubilisation as sulphide makes it possible
to observe the threshold values for discharge; [0080] hexavalent
chromium, which cannot precipitate efficiently as hydroxide without
having been previously reduced to trivalent chromium by the
hydrogen sulphide; and [0081] manganese, zinc and lead, the
precipitation of which as sulphides makes it possible to observe
the threshold values for discharge, unlike the precipitation
thereof as hydroxides.
[0082] Examples of effluents which can be treated according to the
invention are: effluents from the chemical, chemistry-related and
petroleum industries and in particular the coatings and pigment
production industry; effluents from the mineral industry and in
particular the glass industry (high discharge of lead); effluents
from the engineering and surface treatment sector; effluents from
the iron and steel and metallurgical sector (in particular for
arsenic, chromium VI, lead and manganese discharges); and effluents
from the waste materials treatment sector.
[0083] In the said process for the decontamination of an effluent
containing one or more dissolved metals according to the invention,
the hydrogen sulphide production stage can be carried out in any of
the manners described above.
[0084] According to a preferred implementation of the said process
for the decontamination of an effluent containing one or more
dissolved metals, the stage of contacting the effluent with the
hydrogen sulphide is carried out at a neutral or basic pH.
[0085] This implementation makes it possible to minimise any
release of gaseous hydrogen sulphide during the contacting stage
and enables more complete precipitation of the metal sulphides,
which are less soluble at a high pH than at a low pH.
[0086] Advantageously, in the said process for the decontamination
of an effluent containing one or more dissolved metals, the
production of hydrogen sulphide and the contacting of the effluent
with the hydrogen sulphide are carried out in separate tanks, in
particular respectively a culturing tank and a reaction tank, the
said process comprising an intermediate stage between the hydrogen
sulphide production stage and the stage of contacting the effluent
with the hydrogen sulphide, the said intermediate stage consisting
in the injection of all or part of the hydrogen sulphide produced
in the culturing tank into the reaction tank.
[0087] At the decontamination stage, in other words the contacting
of the polluted effluent and all or part of the hydrogen sulphide
produced, in the reaction tank, or after that said stage, it is
possible to add coagulating agents such as FeCl.sub.3, in order to
cause flocculation of the insolubilised metal sulphides, then if
necessary to pass the effluent into a lamellar decanter in order to
separate the precipitates after sedimentation.
[0088] According to a particular implementation of the process for
the decontamination of an effluent containing one or more dissolved
metals according to the invention, a fraction of the effluent is
injected into the culturing tank.
[0089] This implementation can be described as production "with
contacting" or "with partial contacting" of the effluent. In cases
where the composition of the effluent is such that the effluent
does not significantly reduce the capacity for the production of
hydrogen sulphide by the bacteria when these are cultured in
contact with that effluent, this implementation can present an
economic advantage.
[0090] According to another particularly preferred, implementation
of the process for the decontamination of an effluent containing
one or more dissolved metals according to the invention, no
fraction of the effluent is injected into the culturing tank.
[0091] This implementation can be described as production "in
parallel with" the effluent. It is particularly advantageous, since
the bacteria destined to produce the hydrogen sulphide never come
into contact with the polluted effluent; now such contacting can
impair the hydrogen sulphide production capacity of the bacteria in
a proportion which is difficult to forecast, depending on their
better or worse resistance to the presence of dissolved metals in
the culture medium.
[0092] Advantageously, the process for the decontamination of an
effluent containing one or more dissolved metals according to the
invention is such that the stage of contacting the effluent with
the hydrogen sulphide in the reaction tank is carried out in the
absence of a gaseous phase, so that there is no release of gaseous
hydrogen sulphide.
[0093] By "in the absence of a gaseous phase" is meant: in the
absence of any contact with air or any other gas. According to the
established terminology, this contacting therefore takes place in a
"flooded medium". This characteristic is advantageous inasmuch as a
release of gaseous hydrogen sulphide is to be expected in the
presence of a gaseous phase, and all the more since the pH in the
reaction tank can be basic but also neutral or acidic, in which
case the chemical equilibrium between dissolved hydrogen sulphide
and gaseous hydrogen sulphide is displaced in favour of the
latter.
[0094] It should be noted that in the other previously cited
implementation modes of the decontamination process according to
the invention, in case of the presence of a gaseous phase, the
concentration of gaseous hydrogen sulphide possibly released during
the contacting stage is preferably less than 5 mg/m.sup.3.
[0095] As regards the culturing tank, this can contain a gaseous
phase. In that case, the gaseous hydrogen sulphide possibly present
in this gaseous phase can serve to reduce the oxidant compounds
introduced into the medium. In case of gas overpressure, the
gaseous hydrogen sulphide can be neutralised by washing with
caustic soda (NaOH). In any case, the concentration of gaseous
hydrogen sulphide released in the culturing tank is preferably less
than 5 mg/m.sup.3.
[0096] According to a preferred implementation of the invention,
the aforesaid process for the decontamination of an effluent
containing one or more dissolved metals comprises the supplementary
stages of: [0097] measurement of the concentration of hydrogen
sulphide produced in the culturing tank, [0098] estimation of the
concentration of the metals or different metals dissolved in the
effluent to be decontaminated, and [0099] adjustment of the
quantity of solution containing hydrogen sulphide having to be
injected into the reaction tank on the basis of the result of the
said measurement and of the said estimation.
[0100] In other words, according to this implementation it is
possible to use the process for the decontamination of an effluent
according to the invention in various applications, that is to say
it is possible to decontaminate effluents having vary diverse
characteristics in terms of concentrations of dissolved metals, by
a simple and immediate adaptation of the process of the invention.
In fact it suffices to have available a sufficiently large
production of hydrogen sulphide, then to adapt the quantity of
hydrogen sulphide for contacting with the effluent to the nature of
that effluent to be decontaminated.
[0101] Non-limiting examples of metal or metals dissolved in a
polluted effluent that can be treated by means of the invention
are: copper, zinc, arsenic, cadmium, chromium, particularly in its
hexavalent form, tin, manganese, mercury, nickel, and lead.
[0102] Recovery, possibly selective, of the metal-containing
precipitates obtained by the process according to the invention,
with a view to their recycling, is possible.
DESCRIPTION OF FIGURES
[0103] FIG. 1a represents the growth of a culture of
Desulfonatronum lacustre under different culturing conditions. The
x axis shows the time in hours. The y axis shows the optical
density measurement at 580 nm.
[0104] FIG. 1b represents the production of dissolved hydrogen
sulphide during culturing of Desulfonatronum lacustre under
different culturing conditions. The x axis shows the time in hours.
The y axis shows the concentration of HS.sup.- in mM.
[0105] For FIGS. 1a and 1b, the culturing condition components
which vary as follows: F=presence of formate; E=presence of
ethanol; S=presence of sulphate; T=presence of thiosulphate;
YE=presence of yeast extract. The different sets of conditions are
represented on both figures in the following manner: [0106] Symbols
x: E and T; [0107] Symbols .largecircle.: E and S; [0108] Symbols
+: E, T and YE; [0109] Symbols .quadrature.: E, S and YE; [0110]
Heavy continuous line: F and T; [0111] Dashed line: F and S; [0112]
Dotted line: F, T and YE; and [0113] Fine continuous line: F, S and
YE.
[0114] FIG. 2a represents the result of culturing Desulfonatronum
lacustre in a "batch" type reactor at pH 9.5. The change in the
concentration of hydrogen sulphide is represented by a dotted line;
that of the concentration of formate is represented as a continuous
line with .largecircle. symbols and that of the optical density at
580 nm (indicative of the concentration of bacteria) is represented
as a continuous line with .quadrature. symbols. The x axis
corresponds to the time in hours. The left-hand y axis corresponds
to the concentrations in mM (for hydrogen sulphide and formate);
and the right-hand y axis corresponds to the OD at 580 nm.
[0115] FIG. 2b represents the result of culturing Desulfonatronum
lacustre in a "batch" type reactor at pH 10. The change in the
concentration of hydrogen sulphide is represented by a dotted line;
that of the concentration of formate is represented as a continuous
line with .largecircle. symbols and that of the optical density at
580 nm (indicative of the concentration of bacteria) is represented
as a continuous line with .quadrature. symbols. The x axis
corresponds to the time in hours. The left-hand y axis corresponds
to the concentrations in mM (for hydrogen sulphide and formate);
and the right-hand y axis corresponds to the OD at 580 nm. It
should be noted that at t=92 hrs an injection of concentrated
medium is performed (see corresponding example below).
[0116] FIG. 3 represents the result of a heavy metals precipitation
experiment according to the invention. Photo A represents a sample
of 150 ml of industrial effluent essentially containing dissolved
lead. Photo B represents the same sample immediately after
injection of 5 ml of culture medium obtained after 60 days of
culturing of Desulfonatronum lacustre, containing more than 50 mM
of hydrogen sulphide. Black particles of lead sulphide are
visible.
EXPERIMENTAL SECTION
Bacterial Strains and Culture Media
[0117] Two bacterial strains are studied here: Desulfonatronum
lacustre (pH: 9.5) and Desulfonatrovibrio hydrogenevorans (pH:
9.5). Both strains are cultured at 37.degree. C. The energy sources
(electron donors) tested are ethanol and formate and the electron
acceptors tested are sulphate and thiosulphate. The strains are
described in detail in the examples below. The selection of the
strains was performed using 5 ml cultures in Hungate tubes.
[0118] The composition of the culture medium (called MLF) is as
follows (in g/L):
TABLE-US-00001 NH.sub.4Cl 1 K.sub.2HPO.sub.4 0.3 KH.sub.2PO.sub.4
0.3 CaCl.sub.2.cndot.2H.sub.2O 0.1 KCl 0.1
MgCl.sub.2.cndot.6H.sub.2O 0.1 Cysteine 0.5 Na.sub.2S 0.04% Widdel
trace elements 1 mL Yeast extract 0.1
[0119] The sodium sulphide, the energy source, the electron
acceptor and the buffer defined below are added to the medium just
before the inoculation of the latter. The sodium sulphide makes it
possible greatly to diminish the redox potential of the medium,
thus creating conditions favourable to the growth of strictly
anaerobic bacteria.
[0120] The buffer for the culturing performed at pH 9.5 is 1.6%
Na.sub.2CO.sub.3.
[0121] Fermentation Device
[0122] The hydrogen sulphide production tests were performed by
culturing of the selected strain in suspension in a "batch" type
reactor (CHEMAP AG, Switzerland) of 20 L capacity.
[0123] The pH, temperature and stirring speed are regulated in a
control box adjacent to the fermenter (reactor). The pH is
continuously regulated by means of a pH probe immersed in the
culture medium. When a pH value lower than the specified point is
detected, a volume of caustic soda or carbonate solution is
injected into the fermenter by means of a peristaltic pump.
[0124] The temperature of the fermenter (37.degree. C.) is
regulated by circulation of thermostatted water through the metal
body of the apparatus.
[0125] The stirring (15 revolutions/minute) is carried out by a
marine type propeller.
[0126] The nitrogen feed is regulated by a ball flowmeter. A slight
overpressure is maintained in the fermenter by the incoming
nitrogen flow and by immersion of the gas outlet in a 1N solution
of caustic soda. This caustic soda solution also makes it possible
to neutralise gaseous effluxes of H.sub.25 that may be emitted
during the fermentation.
[0127] A tap located at the bottom of the reactor tank makes it
possible to take samples during the fermentation.
[0128] To start this, the reactor, containing 10 litres of MLF
culture medium and the thiosulphate, is initially sterilised with
steam then cooled under a current of nitrogen. The formate and an
inoculum of 2 litres of a culture at the end of the exponential
growth phase are then injected. The pH is then adjusted by addition
of sterile 40 mM carbonate and deoxygenated.
[0129] Thereafter, the compounds added to the culture (caustic
soda, carbonate, culture MLF medium) are not sterilised or
deaerated beforehand.
[0130] Monitoring of Growth
[0131] The growth of the bacteria is monitored by means of a
spectrophotometer by measurement of the optical density of the
cultures at a wavelength of 580 nm.
[0132] Previously performed studies made it possible to establish a
linear relationship between the cell concentration of the bacterium
Thermotoga elfii (g of dry weight/litre), a thiosulphate-reducing
bacterium, and the optical density at 580 nm when the latter does
not exceed 0.8: [Cells]=0.73.times.OD.sub.580 nm. This method is
therefore applied here for the other bacteria.
[0133] The presence of precipitates in the cultures of
Desulfonatronum lacustre imposes a mineralization of these
necessary prior to the measurement of the optical density. This
mineralization is carried out by addition of 400 .mu.L of 1M
persulphuric acid to 4 mL of bacterial culture. After stirring, the
mixture is allowed to stand for 2 minutes before reading of the
optical density at 580 nm.
[0134] Colorimetric Assay of Sulphide
[0135] The dosage of dissolved sulphide is performed according to
the method of Cord Ruwish (Cord Ruwish R., J. Microbiol. Methods.
4: 33-36, 1985): after sampling of 0.1 mL of culture medium, this
is rapidly mixed by vortexing with 4 mL of 5 mM CuSO.sub.4, 50 mM
HCl. The Bordeaux red-coloured copper sulphide thus formed is
titrated by measurement of the absorption at 480 nm and comparison
with a pre-recorded standard curve.
[0136] The dosage of total sulphide is performed by prior
basification of the culture medium to pH 12. This results in the
dissolution of all of the sulphide which is then titrated as stated
above.
[0137] Colorimetric Assay of Thiosulphate
[0138] The dosage of thiosulphate is performed according to the
method described by Nor & Tabatabai (Nor Y. M.sctn. and
Tabatabai M. A., Anal. Lett. 8: 537-547, 1975): 1 mL of sample is
mixed with 1 ml of 0.1M KCN.
S.sub.2O.sub.3.sup.2-.fwdarw.SO.sub.3.sup.2-+CNS.sup.-
[0139] After 15 minutes, 2 mL of 0.33 M CuCl.sub.2 then 1 mL of
Fe(NO.sub.3).sub.3--HNO.sub.3 are added.
CNS.sup.-+Fe.sup.3+.fwdarw.Fe--CNS
[0140] The mixture is adjusted to 25 mL with osmosed water, then,
after stirring and two minutes wait time, the quantity of sulphur
present in the sample is determined by measurement of the
absorption of the iron-thiocyanate complex at 460 nm and comparison
with a pre-recorded standard curve.
[0141] Dosage of Sulphate
[0142] The sulphate is titrated according to the method of
Tabatabai (Tabatabai M. A., Sulfur Institut Journal 10: 11-13,
1974): 1 mL of sample is withdrawn under sterile conditions then
acidified with 250 ml of 1N HCl in an Eppendorf tube in order to
remove sulphides. The cells are removed by centrifugation (14000
rpm, 3 minutes) and 0.5 mL of supernatant is taken up in 4.5 mL of
distilled water. Addition of 250 .mu.L of BaCl.sub.2 (1%
w/v)-gelatine (0.3% w/v) makes it possible to precipitate the
sulphate in the form of barium sulphate. After standing for 30
minutes, the mixture is homogenised on the vortex and the
absorption is measured at 420 nm.
[0143] The standardisation is performed following the same protocol
with 5 mL of distilled water and 250 .mu.L of BaCl.sub.2-gelatine
solution.
[0144] The calibration is carried out on the basis of sulphate
standards (1 mM, 5 mM, 10 mM and 20 mM Na.sub.2SO.sub.4) to which
the above protocol is applied.
[0145] Dosage of Sugars, Organic Acids and Alcohols
[0146] The dosage of soluble metabolic products is performed by
separation by high-pressure liquid phase chromatography (HPLC) on
an ORH801 column (Interaction chemicals) with detection with an RID
6A differential refractometer (Shimadzu). The eluent is a filtered
(0.65 .mu.m Millipore) 0.005N H.sub.2SO.sub.4 solution.
[0147] The samples are centrifuged (1300
revolutions.min.sup.-1.times.15 min) before being injected.
[0148] Dosage of Gases
[0149] The dosage of gaseous products of the bacterial metabolism
is performed by gas phase chromatography (GPC). The chromatograph
(Chrompack CP 9000) is equipped with two columns mounted in series
(Silicagel GC and Molecular Sieve 5A). Detection is carried out by
thermal conductivity using a catharometer. The carrier gas is
helium at 2 bars, and the detector filament current is 70 mA. The
gas injections (0.1 mL) are performed using a syringe fitted with a
valve of the Minimert.TM. type making it possible to maintain the
pressure of the sample.
EXAMPLES
Description of Desulfonatronum lacustre
[0150] This is a sulphate- and thiosulphate-reducing, alkaliphilic,
salt-tolerant and chemolithotrophic bacterium.
[0151] Morphology: vibrio, 0.7-0.9.times.2-3 .mu.m, isolated, in
pairs or in spiral chains, mobile by means of a polar flagellum.
Gram negative. Multiplication by binary fission. Colony in agar:
lenticular, 0.5-2 mm O, yellowish then brown, translucent, with
whole edge.
[0152] Metabolism: non-fermentative
[0153] Electron acceptors: sulphate, sulphite, thiosulphate.
[0154] Dismutation of thiosulphate to sulphide+sulphate.
[0155] Electron donors: H.sub.2--CO.sub.2, formate,
ethanol.fwdarw.acetate.
[0156] No growth on acetate, propionate, butyrate, pyruvate,
lactate, malate, fumarate, succinate, methanol, glycerol, choline,
betaine, casamino acids, yeast extract, glucose, fructose, mannose,
xylose or rhamnose.
[0157] Syntrophy with Spirochaeta alcalica or Desulfonatronovibrio
hydrogenovorans.
[0158] Presence of cyt c, absence of desulphoviridine.
[0159] Growth stimulated by yeast extract or acetate.
[0160] Physiology: T optimum=37-40.degree. C. (20-45.degree. C.).
[0161] pH optimum=9.5 (8-10). [0162] NaCl optimum=0% w/v (0-10%
w/v).
[0163] Dependent on sodium ions and carbonate.
[0164] DNA: 57.3 mol % G+C
[0165] RNA 16S sequence: Y14594
[0166] Typical strain Z-7951 (DSM 10312).
[0167] Origin: sediment from Lake Khadyn.
[0168] Reference: Pikuta E V, Zhilina T N, Zavarzin G A, Kostrikina
N A, Osipov G A, Rainey F A (1998) Desulfonatronum lacustre gen.
nov., sp. nov., a new alkaliphilic sulphate-reducing bacterium
utilizing ethanol. Microbiology (Engl. Tr. Mikrobiologiya) 67,
123-131.
[0169] Description of Desulfonatrovibrio hydrogenevorans
[0170] Sulphate- and thiosulphate-reducing, alkaliphilic, weakly
halophilic bacterium.
[0171] Morphology: vibrio, 0.5.times.1.5-2 .mu.m, 1 polar
flagellum, filamentous appendages, isolated or in pairs or in short
chains, Gram negative.
[0172] Metabolism: lithoheterotrophic
[0173] Electron acceptors: sulphate, sulphite, thiosulphate.
[0174] Electron donors: H.sub.2+CO.sub.2, formate.
[0175] Carbon sources: acetate with yeast extract or vitamins.
[0176] Formation of sulphide on dimethyl sulphoxide with no
growth.
[0177] Growth inhibited by sulphur.
[0178] Disproportionation of thiosulphate to sulphate and
sulphide.
[0179] No desulphoviridine.
[0180] Physiology: alkaliphilic weakly halophilic
[0181] T optimum: 37.degree. C. (15-43.degree. C.)
[0182] pH optimum: 9.5-9.7 (8-10.2)
[0183] NaCl optimum: 3% (1-12%)
[0184] t.sub.1/2 optimum=26.5 hrs on sulphate, 20.1 hr on
thiosulphate.
[0185] Dependent on Na.sup.+
[0186] DNA: 48.6 mol % G+C (Tm)
[0187] RNA 16S sequence: X99234
[0188] Typical strain: Z-7935T (DSM 9292 T).
[0189] Origin: sediments of alkaline lakes (equatorial Lake
Magadi)
[0190] Reference: Zhilina T N, Zavarzin G A, Rainey F A, Pikuta E
N, Osipov G A, Kostrikina N A (1997) Desulfonatronovibrio
hydrogenovorans gen. nov., sp. nov., an alkaliphilic,
sulphate-reducing bacterium. Int. J. Syst. Bacteriol. 47,
144-149.
[0191] Determination of the Optimal Culture Conditions for the
Production of Hydrogen Sulphide by Desulfonatronum lacustre
[0192] Reference is made here to FIG. 1a, which represents the
measurement of the optical density with the passage of time, which
is directly related to the concentration of the bacteria, as a
function of time; and to FIG. 1b, which represents the measurement
of the concentration of dissolved hydrogen sulphide as a function
of time.
[0193] Different culture conditions are tested:
[0194] F: presence of formate; E: presence of ethanol; S: presence
of sulphate; T: presence of thiosulphate; YE: presence of yeast
extract (Panreac, Spain).
[0195] The experiments were performed in duplicate. FIGS. 1a and 1b
represent the mean of the results derived from the measurements of
the OD at 580 nm and of the hydrogen sulphide dosage for the
different cultures.
[0196] Examination of these results shows that: [0197] the use of
formate makes it possible to obtain higher concentrations of
hydrogen sulphide than the use of ethanol; [0198] cultures in the
presence of thiosulphate make it possible to obtain higher
concentrations of hydrogen sulphide than in the presence of
sulphate; [0199] the addition of yeast extract to the culture
medium does not make it possible to obtain results significantly
different from those obtained without that addition; the reason for
this is that yeast extract is present in trace amounts in the
inoculum of bacteria; [0200] Desulfonatronum lacustre reduces
thiosulphate by oxidation of formate producing high concentrations
(about 22 mM) of hydrogen sulphide; [0201] the rate of production
of hydrogen sulphide is 0.122 mM/hr.
[0202] Desulfonatronum lacustre is thus suitable for the production
of hydrogen sulphide and was retained for the continuation of the
experiments owing to: [0203] its ability to produce high
concentrations of hydrogen sulphide; [0204] its rate of production
of hydrogen sulphide; [0205] its alkaliphilia enabling greater
solubility of the hydrogen sulphide and a selection pressure for
maintenance of the strain in culture. The high pH values eliminate
exogenous contamination problems.
[0206] Production of Hydrogen Sulphide by Desulfonatronum lacustre
in "Batch" Reactor
[0207] 1. Importance of Carbonate for Growth
[0208] The implementation of the first cultures of Desulfonatronum
lacustre in a batch reactor made it possible to confirm the
dependence of this strain on carbonate. In fact, in the absence of
sodium carbonate in the culture medium, no growth of this bacterium
was observed for a week. The addition of a concentration of sodium
bicarbonate less than 16.6 g/l does not enable growth either. It
has been postulated that carbonate is involved in the dissociation
equilibrium of formate via the CO.sub.2 concentration of the
gaseous phase.
[0209] 2. Growth and Production at pH 9.5
[0210] A culture of Desulfonatronum lacustre was carried out in the
first instance by inoculation of 10 litres of medium with an
inoculum consisting of two litres of a culture at the end of the
exponential growth phase. The energy source is represented by
formate (74 mM) and the terminal electron acceptor is thiosulphate
(20 mM). The adjustment of the pH to 9.5 is initially performed
with sodium carbonate. The pH is then regulated by injection of 4N
caustic soda solution. The results of the experiment are shown on
FIG. 2a.
[0211] The growth of Desulfonatronum lacustre follows a typical
curve of sigmoidal shape: after a latency phase (t<50 hrs), the
bacteria enter a phase of exponential growth (50
hrs.ltoreq.t.ltoreq.200 hrs), then retardation phase appears
(t>200 hrs). A stationary phase is observed from t=200 hrs. The
formate being monitored in real time, the energy source was
replenished for a second series of experiments at pH 9.5 after its
concentration reached 0 mM.
[0212] The optical density of the culture is remarkably low and
only increases very slightly on addition of formate: a growth
limitation seems to be in operation. The generation time of
Desulfonatronum lacustre under the experimental conditions is 71.45
hrs at pH 9.5. This value is remarkable, even for a
thiosulphate-reducing bacterium. By comparison, the generation time
of Desulfonatronum lacustre under the same experimental conditions
(medium, formate, thiosulphate, yeast extract, temperature, pH) but
during its culturing in a Hungate tube (5 mL of medium) is 84.52
hrs. It thus appears that culturing in a fermenter where stirring
and continuous regulation of the pH are maintained and where the
liquid volume/gas volume ratio is higher makes it possible to
decrease the generation time of this bacterium.
[0213] Observation of the consumption of formate shows that this is
correlated with the bacterial growth. Low during the initial
latency phase, the consumption of formate increases during the
exponential growth phase then slows during the stationary phase.
However, it is noted that the consumption of formate is maintained,
which is consistent with the observation of the maintenance of
hydrogen sulphide production activity by the bacteria in the
stationary state.
[0214] By correlation between the quantity of formate consumed and
that of hydrogen sulphide produced, it is deduced that under the
experimental conditions the formation of one mole of hydrogen
sulphide requires the oxidation of 2.83 moles of formate at pH
9.5.
[0215] After establishment of a biomass inside the reactor and
exhaustion of the energy source, a correlation was established
between the optical density of the culture at 580 nm and the
biomass. Thus for an OD.sub.580 nm of 0.465, a dry weight of 67.6
mg/L was determined. Assuming that the relationship
biomass=ft(OD.sub.580 nm) is linear, we have:
Biomass (mg/L)=145.37.times.OD.sub.580 nm
[0216] It was possible to determine the specific rate of production
of hydrogen sulphide. The specific rate of production of hydrogen
sulphide at pH 9.5 under the experimental conditions is 3.318 mmol
HS.sup.- g.sup.-1 hr.sup.-1.
[0217] 3. Growth and Production at pH 10
[0218] Growth of Desulfonatronum lacustre was then carried out at
pH 10 by addition of concentrated culture medium to the reactor and
alteration of the specified value for the pH. The results of this
experiment are presented in FIG. 2b. At t=92 hrs, the culture
medium was enriched by addition of 1 L of concentrated culture
medium (equivalent to 8 litres of standard medium) in order to
replenish the energy source the concentration of which was
decreasing markedly. This addition caused a decrease in the
concentration of hydrogen sulphide and a decrease in the OD.sub.580
nm.
[0219] It can be observed that the growth of Desulfonatronum
lacustre is maintained at pH 10. This is still correlated with the
oxidation of the energy source, the formate. It is however more
than twice as slow as at pH 9.5. In fact the generation time of
Desulfonatronum lacustre under our experimental conditions is
141.45 hrs at pH 10.
[0220] It can be noted that the concentration of hydrogen sulphide
can attain the value of 32 mM. This high concentration of sulphide
can be the cause of the decrease in the rate of growth of
Desulfonatronum lacustre. This strain can however be considered
exceptionally tolerant to sulphides in view of the concentrations
of hydrogen sulphide encountered in the culture medium.
[0221] By correlation between the quantity of formate consumed and
that of hydrogen sulphide produced, we deduce that increasing the
pH of the culture medium to a value of 10 does not change the
energy yield of the formation of hydrogen sulphide. In fact, the
values at pH 9.5 and pH 10 are almost identical: the formation of
one mole of hydrogen sulphide requires the oxidation of 2.66 moles
of formate at pH 10.
[0222] However, the specific rate of production of hydrogen
sulphide decreases slightly (11%) with the change in the pH from
9.5 to 10. The specific rate of production of hydrogen sulphide at
pH 10 under our experimental conditions is 2.974.+-.0.635 mmol
HS.sup.- g.sup.-1 hr.sup.-1.
[0223] This decrease in the activity of Desulfonatronum lacustre
with the change from pH 9.5 to pH 10 is not in agreement with the
results of Pikuta et al. who observed a 50% decrease in the
sulphidogenic activity of this strain with the change from pH 9.5
to pH 10.
[0224] During culturing performed at pH 10, the medium added to the
reactor was neither sterilised nor deaerated. Microscopic
examination made it possible to check that only Desulfonatronum
lacustre was able to maintain itself under its growth-restrictive
conditions (high pH, inorganic medium, high concentration of
sulphides). Further, the high concentration of sulphides in the
reactor makes it possible to maintain complete absence of oxygen
therein, as is shown by the maintenance of a high concentration of
hydrogen sulphide.
[0225] Optimisation of the Culture Medium
[0226] Cultures were performed in Hungate tubes in different
culture media derived from the standard culture medium MLF.
Depending on the culture series, the cysteine, the sodium sulphide,
the acetate, the yeast extract or the trace elements were omitted.
The first inoculation of Desulfonatronum lacustre was performed
from a pre-culture on rich MLF medium. Thereafter, for the second
(t=10 days) and third (t=20 days) subcultures, the inoculum was
provided by the culture previously performed on the same type of
medium. These subcultures in series make it possible to ensure the
absence of the desired compound through the effect of the
dilutions.
[0227] It should be recalled that in the medium MLF, the cysteine
and the sodium sulphide are added as reducing agents making it
possible to favour the establishment of a low redox potential
favourable to the growth of Desulfonatronum lacustre. The acetate
is added to provide a complementary source of carbon readily
available to the bacterial strain, which should allow it to
initiate its growth. The yeast extract is a source of amino acids
and of inorganic growth factors for this heterotrophic strain and
the solution of Widdel trace elements provides many metals that may
be involved in the bacterial metabolism.
[0228] The results derived from the third subculturing of the
different alternative culture media are shown in Table 1 below.
From the comparison of these results, it can be noted that the
three lowest growths and the three lowest hydrogen sulphide
production levels are correlated with the absence of yeast extract
(media B, D and I).
[0229] A low concentration of yeast extract (0.1 g/L) appears
indispensable for good growth of Desulfonatronum lacustre and high
production of hydrogen sulphide by this strain. To a lesser extent,
the omission of the solution of Widdel trace elements appears to
limit the production of hydrogen sulphide by Desulfonatronum
lacustre. The role of these trace elements can be explained by the
presence of metals in the haems of the enzymes involved in the
sulphate respiration in the sulphate-reducing bacteria. These
elements could be supplied by the impurities encountered in
products of industrial quality and which are not found in the
products of "analytical" quality used in the laboratory.
TABLE-US-00002 TABLE 1 Concentrations Compounds (mg/l) A B C D E F
G H I Cysteine 0.5 X X X X X X Sodium sulphide 0.4 X X X X X X
Sodium acetate 0.16 (2 mM) X X X X Yeast extract 0.1 X X X X X X
Widdel 1 ml X X X X X X trace elements Results of 3.sup.rd
OD.sub.580 nm 0.23 0.18 0.25 0.18 0.3 0.22 0.23 0.24 0.2
subculturing* HS.sup.- 9.35 6.45 10.5 2.35 14 10.9 8.85 12.5 1.9
*the results are the mean of two series of experiments.
[0230] Precipitation of Copper by Hydrogen Sulphide
[0231] The dosage of the dissolved hydrogen sulphide is performed
by measurement of the absorbance of the Bordeaux red-coloured
copper sulphide formed by the action of the hydrogen sulphide on
copper sulphate. The reaction involved in this dosage was exploited
to illustrate the capabilities of hydrogen sulphide for the
separation of dissolved metals in an effluent.
[0232] The copper sulphide is formed instantaneously on mixing of
the hydrogen sulphide and the dissolved copper with stirring. The
copper sulphide thus formed precipitates rapidly, in a manner
visible to the naked eye. When the mixture is allowed to stand for
5 minutes, the major part of the precipitate is found at the bottom
of the test-tube.
[0233] The addition of a coagulant such as FeCl.sub.3 makes it
possible to precipitate the whole of the copper. The precipitation
of metals in the form of sulphides by the use of the hydrogen
sulphide present in cultures of sulphate- and thiosulphate-reducing
bacteria has been confirmed.
[0234] Decontamination of an Industrial Effluent
[0235] A precipitation of heavy metals contained in an actual
industrial effluent provided by the company X, situated in the
Vaucluse and confronted with contamination of its effluents with
the lead which it converts in the context of its activity of
accumulator production, is performed.
[0236] Desulfonatronum lacustre is cultured at 37.degree. C. for
more than 2 months in 2 litres of MLF medium. The initial pH of the
culture is 9.5. The energy source utilised is formate, and the
sulphur source is thiosulphate.
[0237] The polluted effluent is drawn off upstream of the currently
existing treatment works.
[0238] A measurement of the concentration of hydrogen sulphide is
first preformed. After dilution of the culture medium to 1/4, the
measurement gives a concentration of 4.times.12.9=51.6 mM. This
exceptional concentration of hydrogen sulphide in a culture medium
is due to a particularly long culturing time (greater than 60
days).
[0239] Analysis of the final pH of the medium shows that this is
8.8. The fall in the pH from 9.5 to 8.8 in spite of the presence of
a carbonate buffer confirms the generation of a large quantity of
acid during the culturing of Desulfonatronum lacustre.
[0240] This concentration of hydrogen sulphide greater than 50 mM
in the culture medium shows that Desulfonatronum lacustre is
capable of tolerating particularly high concentrations of
sulphides.
[0241] 5 ml of culture medium containing 51.6 mM of hydrogen
sulphide are withdrawn by means of a syringe and injected into 150
ml of contaminated effluent. The effluent before injection is shown
on photo A in FIG. 3, and the effluent immediately after injection
is shown on photo B in FIG. 3.
[0242] Following the injection of the hydrogen sulphide into the
effluent, particles of lead sulphides, identified by the
characteristic lustrous black colour, appear and rapidly
sediment.
[0243] The injection of culture medium containing hydrogen sulphide
produced by the metabolism of Desulfonatronum lacustre thus makes
it possible instantaneously to effect the precipitation of the lead
and its separation from the industrial effluent.
* * * * *